Many drug-drug interactions are metabolic in origin, resulting from the activation or inhibition of Cytochrome P450 (CYP)-dependent drug clearance. A potential contribution to CYP-dependent drug interactions is the poorly characterized allostery that is widely observed in vitro, and also in vivo. This allostery includes non-hyperbolic steady state kinetic profiles for individual substrates (homotropic allostery), as well as alterations in the kinetic profiles of one drug by a second (heterotropic allostery). The molecular mechanisms leading to allostery are not defined, although several contrasting models have been proposed and include multiple ligand binding within a single fluid active site, multiple ligand binding within discrete subsites of this large active site, and ligand-dependent persistent protein conformational equilibria. Here, non-steady state kinetic methods will be exploited to challenge these models and to understand CYP allostery in the context of existing structural models for CYP3A4 and CYP2C9. The specific aims are: 1) to determine whether the elementary steps of ligand binding, ligand-dependent ferric spin state equilibrium, or ligand-dependent changes in gross protein conformation are differentially allosteric for the homotropic effects of testosterone, pyrene, and hypericin with CYP3A4; 2) to determine by paramagnetically shifted 1H-NMR spectra, the mechanism of allostery for the heterotropic effects between caffeine and acetaminophen or midazolam and alpha-naptho flavors with CYP3A4, and to initiate SAR-by-NMR to map ligand binding sites in uniformly deuterated, 15N-Phe-labeled CYP3A4; 3) to monitor conformational dynamics of CYP3A4 via limited proteolysis/electrospray mass spectrometry as a function of ferric/ferrous redox state and ligand binding; 4) to map the CYP3A4/Cyt b5 binding surface via site-directed mutagenesis and catatylic turnover experiments, and determine whether specific interfacial residues contribute to Cyt b5-dependent allosteric effects. In addition, where possible, parallel experiments will be performed with CYPeryF, a soluble homolog for which X-ray crystallographic analysis is possible. In the long term, x-ray structures of [CYPeryF.ligand] complexes will be sought, via collaboration, in order to correlate allosteric effects with CYP structure.